The present invention generally relates to a method for diagnosing malaria, and more particularly to a highly specific diagnosis method based on the detection of malaria parasite glycolytic enzymes.
Malaria is one of the most wide-spread infectious diseases. The World Health Organization estimates that 200,000,000 people are infected with the malaria parasite annually. These people mainly reside in the tropics. One million cases of malaria were reported in the United States in 1940. At that time, effective measures were introduced which virtually eliminated the disease, which is transmitted by the female Anopheles mosquito. However, the Anopheles mosquito is still present in many of the Southern and Western parts of the United States. During the early 1970's, there were several cases of malaria reported in Louisiana and California. These were attributed to returning veterans from the Viet Nam war who harbored the parasite.
As tropical regions of the world become more accessible through improved modes of transportation, travel into these areas is increasing. This has resulted in significantly more cases of malaria being reported in travelers returning from these areas. One percent (1%) of all people infected with the malaria parasite die from the disease. Specifically, in 1989, 2,700,000 people died of malaria. There are four species of Plasmodium which infect humans and cause malaria. These include P. falciparum, P. vivax, P. ovale, and P. malariae. P. falciparum is the most serious species. It is responsible for cerebral malaria which is associated with a 50% mortality rate.
There are now over 65 countries which have reported chloroquine-resistant P. falciparum, and 12 countries which have reported quinine-resistant P. falciparum. These two anti-malarial drugs were the major therapeutic agents used to treat the disease, and their indiscriminate use has added to the spread of resistant strains of malaria parasite. Since these drugs are rapidly becoming entirely or partially ineffective for specific parasite species, it is essential that an accurate diagnosis of the disease be obtained in order to provide proper treatment. The diagnostic method of choice must be rapid, specific, readily available, easy to perform, and easy to interpret.
The life cycle of a Plasmodium parasite involves the interrelationship between an Anopheles mosquito vector and a mammalian host. When an uninfected female Anopheles mosquito bites and ingests blood from a host harboring the sexual forms of the Plasmodium parasite, the parasitic life cycle begins. In the Anopheles, the male and female gametocytes fuse and travel after several stages of development to the salivary glands of the mosquito. The parasite at this stage is called a "sporozoite." If the infected mosquito bites a new host, the sporozoites are injected into the host's blood. Thereafter, they travel to the liver within 30 minutes, where they enter a liver cell. In the liver cell, one sporozoite multiplies and forms about 10,000-20,000 merozoites. These merozoites are released from the liver cells in 10-12 days. Each of the released merozoites immediately invades an erythrocyte. In 48 hours, each merozoite forms another 10-12 merozoites which are in turn released from the erythrocyte only to invade another 10-12 erythrocytes.
The clinical manifestations of the disease include fever, headaches, sweating, vomiting, and prostration. These manifestations occur simultaneously with merozoite release from the erythrocytes. The erythrocyte reinvasion occurs until the host dies, or until the host's immune system is able to control and suppress merozoite activity. At some point, the merozoites (previously asexual) differentiate into male and female gametocytes. The technical and scientific basis for this transformation is an active area of current medical research. If a female Anopheles then bites a new host at the time of gametocyte formation, the life cycle of the parasite is completed.
The most susceptible human hosts for the disease are infants and pregnant women having suppressed immunity. Recently, deaths have been reported in adult male AIDS patients caused by cerebral malaria. In addition, non-immune travelers into high-risk malaria areas are also susceptible to the disease, especially with respect to chloroquine and quinine resistant malaria.
There is a natural immunity to malaria which develops in persons living in high-risk malaria areas. This immunity appears to depend upon the continual presence of low parasite levels in the host's body. This conclusion is drawn from many studies which demonstrate that when persons living in high-risk malaria areas leave and travel to low-risk areas, they substantially lose their immunity.
As previously mentioned, the disease must first be properly diagnosed before treatment may be given. Ideal diagnostic methods must be specific, sensitive, accurate, easy to implement, and require a minimum of complex diagnostic equipment. Numerous approaches have been taken regarding the laboratory diagnosis of malaria. These approaches include the use of thick and thin blood smears treated with a conventional stain known as "Giemsa" and examined with a light microscope. Other methods range from fluorescent dyes to recently developed methods involving DNA probes, indirect fluorescent antibody tests, indirect haemagglutination tests, enzyme-linked immunosorbent assays, and gel precipitation tests as extensively discussed in Bulletin of the World Health Organization: Malaria Diagnosis, 1045, 1-37 (1988).
With respect to other malaria diagnostic procedures, U.S. Pat. No. 3,834,874 to Geating et al. involves a Plasmodia detecting apparatus consisting of a pre-stained microscope slide covered with a dried mixture of methylene blue NN and cresyl violet acetate.
Australian patent application 66/04,418 discloses a blood smear composition comprising a solution of methylene blue chloride, an alkali metal bicarbonate (preferably NaHCO.sub.3), eosin Y, and azure (II)-eosin in an alcohol mixture. The resulting composition is designed to detect malaria parasites and may be used for all types of blood smears (thick and thin). A similar process is disclosed in British Patent 1,183,499 which involves a biological stain for detecting malaria in which a blood film is first stained with polychrome methylene blue solution, followed by treatment with an eosin solution.
U.S. Pat. No. 4,741,898 discloses a stabilized Romanowsky-type stain composition for malaria parasites which includes a cationic dye component (methylene blue, azure A, azure B, azure C, or thionin), an anionic dye component (eosin Y, eosin B, fluorescein, substituted fluorescein, or orange G), a 1-6 carbon alcohol solvent, and a stabilizer (e.g. lysine or glycine).
Further information regarding malaria staining techniques is disclosed in Bianco, A. E. et al., "Plasmodium Falciparum: Rapid Quantification of Paracitemia in Fixed Malarial Cultures by Flow Cyctometry", Exp. Parasitol., 62:75-282 (1986); Tanabe, K., "Staining of Plasmodium Yeoli-Infected Mouse Erythrocytes with the Fluorescent Dye Rhodamine 123", J. Protozool., 30:707-710 (1983); Makler, M. T. et al., "Thiazole Orange: A New Dye for Plasmodium Species Analysis", Cytometry, 8:568-570 (1987); and Patton, C. L. et al., "Diagnosis of Malaria Using Quantitative Buffy Coat (QBC.TM.)", Documents Complementaires: Resume of 3rd International Conference on Malaria and Babesiosis, Annecy 1987.
The article by Makler, M. T. et al. is of particular interest in that it discloses a fluorescent staining technique for Plasmodium falciparum using a membrane-permeable fluorochrome thiazole orange dye in conjunction with a fluorescent flow cytometer.
A variety of other diagnosis methods exist for detecting the presence of malaria infections. For example, French patent application 2,572,528 involves a process for identifying, sorting, and counting microscopic particles (including Plasmodia) in which test samples are first prepared and deposited in succession on an advancing conveyor surface. The conveyor surface moves the samples into the viewing field of a microscope providing images which are recorded by a video camera. The recorded images are then set against a squared pattern grid so that the image corresponding to each sample may be analyzed according to standard pattern recognition techniques.
European patent application 119,209 involves a method for identifying microorganisms including malaria parasites through the use of DNA probes. The parasites are first immobilized on a solid support. DNA from the parasite sample is then subjected to hybridization with a labeled specimen of species-specific, non-cross hybridizing DNA from a known species. The parasite sample is then examined for hybridization between the first and second DNA materials.
Canadian Patent 951,242 involves an immunoglobin M diagnostic reagent for detecting malaria and other diseases consisting of polystyrene particles coated with IgM. To determine IgM levels in a sample of test serum and detect disease infestation, the serum is first mixed with human IgM antiserum followed by combination with the polystyrene-IgM reagent. If agglutination occurs, IgM levels are normal and there is no disease.
Australian patent application 87/72,041 discloses a method in which nucleotide sequences from the RESA antigen of Plasmodium falciparum are used as detection probes.
Notwithstanding the methods described above, a need exists for a malaria diagnosis procedure which is easily used, requires minimal amounts of instrumentation, and is parasite sensitive and specific. The method must be economical, and capable of providing accurate test readings in remote geographical regions. The present invention satisfies these goals, and represents an advance in the field of disease diagnosis, as described below.